Table 13 provides a comparison of various assay
procedures for protein quality evaluation. The choice of
procedure is to some extent determined by the various criteria
desired. Thus, some assays are more suitable for screening
because they use a small sample, are relatively inexpensive, have
little complexity, and often provide information on the limiting
amino acid and on complementary potential. Scoring and
digestibility data can require moderately complex analytical
techniques, although in some circumstances published data may be
used for calculation of mixed dietaries, as discussed in chapter
3. The limitation of these procedures, however, is that they have
only moderate capacity for discriminating among proteins of
differing quality. Table 13 also presents the characteristics of
human and rat assays that can be performed at single or multiple
levels The complexity of these techniques, the size of the sample
required, and the expense, preclude their use for screening
purposes. Furthermore, by themselves (i.e., without performing
the assays several times in the presence of added amino acids},
they do not identify limiting amino acids or provide information
on their complementary nature. The values obtained are, however,
inclusive of digestibility and availability and also provide the
ability to recognize the presence of toxins. Their main strength
lies in their ability to discriminate among proteins in the
determination of quality, especially the procedures involving
multiple levels of protein.

TABLE 13. Some Criteria for the Selection of an
Appropriate Protein Assay Procedure

Table 14 describes in more detail individual
rat assays of protein quality. Five of the tests, as have been
described in chapter 4 and summarized in table 8, are performed
at only one protein level with or without a non-protein control
group, whereas the remaining three are multi-level tests. It will
be noted that NPU can be performed in two ways, either by carcass
analysis or by balance technique. If digestibility data are
required, faecal analysis will be necessary in addition to the
carcass analysis in the former procedure for NPU. The eight tests
largely fall into the two categories of single-level and
multi-level when their ability to rank proteins and to
discriminate among proteins is examined. Most single-level tests
are poor or moderately good in these capacities, while the
multi-level tests may be more easily reproduced in different
laboratories. Furthermore, by definition, the single-level assays
cannot provide any indication of linearity of response to dose,
whereas the multi-level assays are constructed to give this
information. The complexity of the testing procedures tends to be
greater for multi-level techniques than for the single-level
procedures except for NPU (balance), which requires urinary and
faecal collection procedures demanding considerable precision.

TABLE 14. Some Criteria for the Selection of an
Appropriate Rat Assay Procedure

Criterion

PER

NPR

RNPR(carcass)

NPU (balance)

NPU

NGI

REV

RPV

A direct
measure of digestibility

no

no

no

no

yes

no

no

no

Rank protein

poor

moderate

good

moderate

moderate

good

good

good

Ability to
discriminate

moderate

moderate

good

moderate

moderate

moderate

good

good

Reproducibility
in other labs

poor

moderate

moderate

moderate

N/A**

moderate

good

good

Applicability
to proteins very low in Iysine

poor

poor

poor

poor

poor

poor

poor

moderate

Proportionality

poor

poor

moderate

moderate

moderate

moderate

moderate

good

Test for
linearity no

no

no

no

no

no

yes

yes

yes

Simplicity

simple

simple

moderate

simple

complex data
collection

moderate

complex
statistical analysis

moderate

Time
required(days)

28

10 - 14

10 - 14

10

9

14

14 - 21

14 - 21

Relative cost

moderate

low

low

moderate

moderate

high

high

high

For proteins extremely low in Iysine, all
bioassay procedures have limitations. This is because, for at
least the period of time used in these assay procedures, rats
have the ability to conserve Iysine, and thus their response is
not directly proportional to Iysine level. The RPV procedure,
which does not include consideration of the zero-protein level,
is better in this respect than the others but is still only
moderately accurate. The length of test period required varies
from 9 to 21 days, except for PER, which, in its official
version, requires 28 days. The cost of these procedures is
generally less for single-level assays than for multi-level ones,
except that the longer period of food consumption at the 9 to 10
per cent level, and the larger number of animals demanded by some
standardized PER procedures, per group, increase costs. It will
be noted that all three of the multilevel procedures tend to be
the most expensive because of the larger number of diets and
animals used.

In summary, PER is the poorest of current
animal tests of protein quality, and if a single-level assay is
chosen, procedures other than PER are preferable on the grounds
that, by using a zero-protein control, they provide a response
that may be more proportional to the quality of the protein. NPU
by balance can be a reliable and precise procedure, especially if
corrected by use of a standard protein. It does, however, require
many analyses of urine and faecal samples and demands
high-quality analytical facilities. If a growth method is to be
used, RNPR would be the recommended procedure because it
incorporates both a zero-protein control group and a standard
protein for comparison. These both increase its reproducibility
and ability to discriminate.

Among multi-level tests of protein quality, RNV
may be more sensitive than the other two because its statistical
design improves its ability to discriminate among proteins. While
RPV is not problem-free for proteins low in Iysine, RNV has more
limitations than RPV in this respect because it includes
non-protein data. In general, the greater complexity and cost of
multi-level assays may not be justified unless analyses are being
performed on sources of protein of low quality, especially those

TABLE 15. Some Criteria for the Selection of an
Appropriate Human Assay Procedure

Criterion

NB

NBI0

RNR*

NVU**

(N B I )

BV

RPV*

A direct
measure of protein digestibility

yes

yes

yes

yes

Rank protein

poor

good

good

good

Applicability
to all proteins regardless of limiting amino acid

poor

good

good

good

Simplicity

moderate

difficult

difficult

difficult

Time required
(days)

14

11 - 50

11 - 50

20

Relative cost

moderate

high

high

high

* This has been termed RPV in some human
studies since the approach is identical. ** At suboptimal intake
of test protein. Note: There are insufficient data to judge the
other criteria listed in table 14 (rat assays) since the methods
used have not been standardized. limited in Iysine, or unless one
is concerned with detecting small differences in quality. In
these circumstances, RNV and NGI may be less reliable than RPV
because of statistical deflection in the slope of the line caused
by the inclusion of the non-protein control group.

For many purposes, the final test of quality
involves evaluation of the protein sources using human subjects
as the consumers. Table 15 compares the merits of four commonly
used procedures. All procedures provide a direct measure of
digestibility because faecal analyses are required. On a limited
intake of the test protein, nitrogen balance by itself is
generally unrewarding; in particular, it has a low capacity for
ranking proteins in order of qua)ity and poor applicability to
proteins that are very low in Iysine. The other three procedures
have a good record for both of these criteria, but they are more
difficult to perform, take a longer time, and are more costly. It
is not currently possible to express a definitive opinion on the
relative values of these three procedures because of the limited
amount of published work on which to make a judgement.

Finally, it is appropriate to summarize the
application of all the preceding tests of protein quality under
the five major circumstances into which the reasons for
assessment of protein quality can be divided.

In general, the systematic study of protein in
any of these circumstances involves initial) chemical testing
followed by biological assays. For the purposes of evaluating
plant protein made available in the course of breeding new
varieties of grains or legumes, the first step is usually
chemical analysis for protein content and amino acid profile.
Sometimes amino acid analysis at this stage may be for a single
critical amino acid, or for a restricted number, such as
tryptophan, sulphur amino acids, and Iysine. If these two
criteria are promising, then the subsequent assays, including
bioassays, are described in detail in several excellent
publications (1-3) that are designed for the express purpose of
following a defined sequence of analyses in circumstances where
the initial sample is often very small. For exploring new sources
of protein, the initial approach is to examine protein
concentration and amino acid pattern from which an amino acid
score can be derived followed by an in vitro test for
digestibility. If these criteria suggest a potentially useful new
protein source, then a bioassay, both to test for availability of
amino acids and as a further test of quality, should be
undertaken. If amino acid pattern and score indicate a
significant limiting amino acid, then the bioassay should be
repeated with the limiting amino acid added to the diet as a
confirmation of the prediction. Finally, tests should be
performed together with another protein source to examine the
complementary potential of the test protein.

These biological tests may reveal a less
favourable picture than indicated by amino acid scoring alone. In
this case, the product should be examined for non-available amino
acids, as for example by use of tests for available Iysine, or
should be evaluated for possible toxic materials present in the
foodstuff. The biological testing should include a measure of
digestibility, because this can be a significant cause of
discrepancy between chemical and biological evaluations of
quality. This could be done either by use of a technique such as
NPU (balance), or by additional determination of faecal nitrogen.

For the third use, namely the monitoring of
variables introduced by food processing, it is common to begin
with availability of Iysine and of methionine, as these are
usually the amino acids most sensitive to processing. If it is
desirable to test the product further, the sequence outlined
above for testing new sources of protein will serve as a
guideline. Many guidelines and statements have been published by
PAG that list in detail the testing sequence required for new
protein products (see appendices A and B2).

For routine regulatory purposes, the
examination of the sample should begin with chemical analysis for
nitrogen, amino acids, and toxins, including microbial toxins.
The regulatory requirements may also indicate bioassays of
protein quality that are commonly. specified in detail by the
regulatory agency but that may not necessarily represent the
choice of the investigator. For example, the customary use in
North America of the PER assay for regulatory purposes has been
highly criticized by many investigators, and represents a failure
to adopt more rigorous procedures.

Finally, tests for protein quality to assess
human protein requirements for such proteins should be conducted.
These need to be performed with the greatest care. In addition to
a knowledge of the amino acid composition of the protein and
relevant animal data, usually originating from multi-level tests,
the examination of nitrogen balance in response to a single level
or several levels of protein is usually the criterion of protein
quality in the assessment of requirements. The merits of
different techniques for measuring nitrogen balance are discussed
earlier and are shown in tables 13 - 15.

These comments are only intended as guidelines
for users not familiar with the detailed literature. It is clear
that many techniques are available and that local circumstances
and availability of facilities, as well as familiarity with the
tests, will determine the final choice. The wide range of costs
of different tests, and the limitations of human assays, may make
it impossible under some circumstances to deploy the complete
battery of appropriate tests outlined above. If the investigator
is indeed restricted by such circumstances, it is nevertheless
hoped that recognition will be given to fact that perhaps only a
partial picture has been obtained.

In conclusion, it must be emphasized that even
when a protein is assayed using the best available procedures,
ancillary studies, both bioligical and chemical, are necessary to
assess fully the potential value of a protein in a real-life
situation. Health, nutritional status, age, and physiological
status of the individual consuming the protein, together with the
complete dietary composition, including other proteins and the
total energy value, all combine and interact to affect the final
value of the protein to the consumer.

The concept of dietary protein quality seems at
first sight to be self-evident. However, upon closer examination,
a precise definition becomes more difficult. The capacity of a
protein source to meet the amino acid and nitrogen requirements
of the organism depends not only upon the amino acid composition
and digestibility of the protein source or mixture, but also upon
the composition and adequacy of the diet as a whole, and on the
physiological, nutritional, and health status of the consumer.
Among the dietary factors that might be included are level and
source of carbohydrate and lipid intake, mineral and vitamin
content, water intake, and size and frequency of meal ingestion.
Nutritional status, age, and health factors interact in a complex
way to modify the utilization of dietary protein. Studies of the
effects of these various factors and their interactions are
necessary in order to define adequately the variability and
nutritional significance of dietary protein quality. However, the
following suggestions for research are readily apparent.

Standard Proteins in Biological Assays

The biological assessment of protein quality
depends upon an appropriate comparison with a suitable reference
protein. In the past, casein has been used as the standard or
"reference protein," and standards of protein quality
in foods have been related most frequently to the nutritive value
of casein. However, there are a number of reasons why casein does
not provide the ideal reference standard. First, due to its
limiting concentration of sulphur amino acids, it is not as
efficiently utilized in meeting the nutritional needs of the rat,
in comparison with proteins such as lactalbumin. Thus, it would
appear more desirable to utiiize a protein of higher quality for
assay purposes. Another difficulty is that even the high-nitrogen
ANRC casein may not be as well standardized a product of constant
nutritional quality as is commonly assumed. Therefore, it is
recommended that research be focused on the development and
standardization of a suitable reference protein. A defined
mixture of L-amino acids should be explored as a possible
reference protein of standard nutritive value. While lactalbumin
has been used in slope-assay procedures, its availability and
lack of standardization do not make this protein an ideal
standard protein at the present time. ANRC casein supplemented
with 1 per cent DL-methionine has proved to be of high
nutritional value in the rat and is recommended for further
evaluation as a possible standard protein for comparative
purposes.

Choice of Animal Species in Bioassay

Bioassay procedures utilizing the laboratory
rat require amounts of test protein that may be in excess of the
amount readily available. Thus, it would be worthwhile to explore
the use of other species for protein quality estimations. Another
problem is the relevance of findings in the rat, or any other
species, to quantitative aspects of protein quality in human
subjects. Although some evidence indicates, as discussed earlier,
that there is a close and quantitative relationship between
studies in growing rats and nitrogen-balance indices in children
who have recovered from malnutrition, the data are limited and it
is not known whether results in children who have always been
healthy would give similar close agreement. Furthermore, there
are insufficient data in the literature to provide a critical
comparison of protein quality in humans of different ages,
although it is assumed that protein quality is of lesser
significance in the adult. In order to develop appropriate
bioassay procedures in rats that have relevance to human
nutrition, these problems must be explored.

Assay Procedures

It might also be questioned whether any one of
the common bioassay procedures is adequate or significantly
better than other assays for all protein sources irrespective of
the limiting amino acid. A particularly difficult problem is the
appropriate choice of assay procedure with rats in the case of
low-quality protein sources, especial)y those limited in Iysine.
Further critical examination of this problem is warranted.

Plasma Amino Acids

The earlier studies relating plasma amino acid
levels and protein quality were carried out without full
appreciation of all the various factors affecting plasma amino
acid levels. With current information it may be possible to
develop a more reliable and rapid protein quality assay with
growing rats, using changes in plasma amino acid patterns. This
approach may also be of potential value for the clinical
evaluation of protein quality. Thus, it would be desirable to
explore critically those experimental conditions under which the
measurement of plasma amino acid levels would provide useful
predictive data on protein nutritional quality in human feeding.
This may be accomplished initially through examination of the
plasma amino acid levels at the termination of standard rat
bioassay procedures. An index of protein quality and
identification of the limiting amino acid might be obtained
through this approach.

Rapid Procedures

With the greater requirement for protein
quality data for use in food labelling and other regulatory
purposes, there is a tremendous need by both the food industry
and the regulatory agencies for improved, rapid but reliable
tests of protein quality. Animal bioassays are time-consuming and
expensive, and they are not ideal for meeting the needs of the
food industry for nutrition labelling of food products. The ideal
test would be reproducible, rapid, inexpensive, and applicable to
a wide variety of food products. Perhaps such a single test is
unattainable. Because protein quality is not dependent on a
single factor but rather is the result of an interaction of a
complex set of variables and interrelated factors, more than one
test may be needed. Such rapid tests should perhaps be concerned
with the separate major components of quality - i.e., amino acid
composition (score), processing damage, digestibility, and amino
acid retention and utilization. Criteria might be devised for
each of these categories rather than for a single quality
measurement.

Finally, there is still a need to undertake
collaborative studies of protein quality assay procedures,
ranging from chemical and in vitro studies to rat and human
bioassays. These collaborative investigations should involve
laboratories both in developed and developing countries. It is
hoped that the United Nations University will take the initiative
for this research effort.